Effects of crude oil/dispersant mixture and dispersant components on PPAR[gamma] activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen.

The Deepwater Horizon (DWH) oil spill, which began 20 April 2010,
resulted in the release of > 200 million gallons of MC252
(Mississippi Canyon block 252) crude oil into the Gulf of Mexico.
Approximately 2 million gallons of dispersant was used to emulsify the
oil into the water column, with the aim of aiding in oil biodegradation
and preventing the oil from reaching fragile near-shore habitats
(Kujawinski et al. 2011). The dispersant applied by aerial spray and at
the wellhead oil source was primarily COREXIT 9500 (EC9500, EC9500A, and
EC9500B) and secondarily COREXIT 9527 (Nalco Environmental Solutions).
Dispersant has been shown to increase the bioavailability of oil
components, such as polycyclic aromatic hydrocarbons (PAHs) to fishes
(Ramachandran et al. 2004). Although many studies in a variety of animal
models have focused on the toxicity of crude oil, dispersed oil, or
dispersant alone (Almeda et al. 2013; Hemmer et al. 2011; Rico-Martinez
et al. 2013), long-term sub-lethal studies are limited.

Both oil and dispersant are implicated as potential endocrine and
metabolic disruptors. Crude oil is linked to reproductive effects in
male rats (Adedara et al. 2014), and dispersants have been shown to be
estrogenic in an in vitro transactivation assay using a human liver
hepatoma cell line (Judson et al. 2010). Also, maternal exposure to PAHs
(major components of oil) in ambient air during pregnancy was associated
with a higher prevalence of obesity at 5 and 7 years of age among
participants in a New York City birth cohort (Rundle et al. 2012). Given
the massive quantity of oil released and the unprecedented use of
dispersant during the DWH oil spill, it is important to understand
potential impacts to human health through direct and indirect exposures
including those resulting from relief efforts and seafood consumption,
respectively.

Obesity is a major health problem that contributes to a variety of
diseases, including type II diabetes, hypertension, and cancer (Calle
2007; Ogden et al. 2006). Although traditionally attributed solely to an
imbalance in energy intake versus expenditure, recent evidence
implicates environmental agents known as "obesogens" as
potential contributors to the obesity epidemic, especially in children
(Grun and Blumberg 2006). The mechanism of action of obesogens is not
completely understood, but any chemical that affects food intake, energy
expenditure, lipid metabolism, or adipocyte (fat cell) differentiation
could potentially act as an obesogen. The master regulator of adipocyte
differentiation, the nuclear receptor PPAR[gamma] (peroxisome
proliferator-activated receptor gamma) (Janesick and Blumberg 2011), is
a prime target on which obesogens act. Obesogens include
diethylstilbestrol (Newbold et al. 2005), bisphenol A (Somm et al.
2009), various phthalates and their metabolites (Stahlhut et al. 2007),
fungicides and insecticides such as triflumizole (Li et al. 2012),
hydrocarbons (Tracey et al. 2013), and the marine anti-fouling agent
tributyltin (Kirchner et al. 2010). It is therefore relevant to
determine whether novel obesogens exist in oil or dispersants.

We investigated the obesogenic potential of COREXIT 9500-dispersed
MC252 crude oil and identified the major COREXIT component, dioctyl
sodium sulfosuccinate (DOSS), as a likely obesogen. In addition to it
being a major component of the dispersant COREXIT, DOSS is widely used
in pharmaceuticals and personal care products [U.S. Department of Health
and Human Services (DHHS) 2014; Environmental Working Group (EWG)
2015a].

Materials and Methods

Transfection and PPAR[gamma] reporter gene assays. For PPAR[gamma]
transactivation assays, HEK293T/17 cells (ATCC CRL-11268) were
maintained in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient
Mixture F-12; Gibco by Life Technologies) containing 10% fetal bovine
serum (ThermoScientific), 2 mM glutamax, 100 [micro]M nonessential amino
acids, and antibiotic/antimycotic. Cells were transfected with
Lipofectamine 2000 according to the manufacturer's protocol
(Invitrogen by Life Technologies) and plated in 96-well dishes at a
density of 20,000 cells per well. Each well of cells was transfected
with 16 ng PPAR[gamma]-Gal4 [fusion protein of the yeast GAL4
DNA-binding domain (amino acids 1-147) and the mouse PPAR[gamma]
ligand-binding domain (a.a. 163-475); kindly provided by B. Blumberg,
University of California, Irvine], 80 ng UASx4 TK-luc [contains four
copies of the GAL4 upstream activating sequence and the herpes virus
thymidine kinase promoter (-105/+51) driving firefly luciferase] and 4
ng of pRL vector (encoding Renilla luciferase to control for
transfection efficiency). The next day, triplicate wells of cells were
treated as described. Cell lysates were harvested after 18 hr of
treatment and the Dual-Luciferase Reporter Assay System (Promega) and a
Veritas microplate luminometer (Turner Biosystems) were used to measure
firefly and Renilla luminescence according to the manufacturers'
recommendations. For assays using human PPAR[gamma], PPAR[alpha], and
PPAR[beta]/[delta] plasmids were kindly provided by B. Abbott, U.S.
Environmental Protection Agency, and are described by Bility et al.
(2004). The GAL4-RXR[alpha] plasmid was kindly provided by B. Blumberg
(Grun and Blumberg 2006).

Solid-phase extraction (SPE). Fractionation of CWAF was performed
to determine if compounds from COREXIT or oil were responsible for the
observed PPAR[gamma] activity. Bond Elut 3-mL silica solid phase columns
(Agilent Technologies) and vacuum manifold chambers were employed to
fractionate CWAF. Four fractions bearing differential polarities and
hydrophobicities were collected in the following order: 50:50
water:ethanol, methanol, dichloromethane, and hexanes. For every 75
[micro]L of CWAF loaded into the column, 100 [micro]L of water was used
to pull the sample through the column, and 2 mL of solvent was used to
collect each fraction. Before downstream applications solvents were
removed by vaporization using a Savant ISS110 SpeedVac Concentrator
(Thermo Scientific) and resuspended in 10 [micro]L DMSO/500 [micro]L of
fraction.

To confirm the presence of Tween 80, the sub-fraction was analyzed
in positive FS mode, revealing several unique mass profiles with mass
differences (between adjacent ions) of 22 and 44 amu, reported to be [[M
+ N[H.sub.4]].sup.+] and [[M + 2N[H.sub.4]].sup.2+] ions of polysorbate
species (Zhang et al. 2012). Product ion scans showed that the m/z 309.3
ion was the most frequent fragment for the target masses. Previous
reports attributed the m/z 309 fragment ion to an in-source loss of
specific fatty acid esters (ethyl oleate, for m/z 309.3) characteristic
of polysorbate species (Hvattum et al. 2012), or more recently to a
strong presence of oleate-related species in polysorbate 80 (Tween 80)
(Zhang et al. 2012). PIS of m/z 309.3 demonstrated several mass spectral
profiles relating to sorbitan monooleates (specifically 16-27
polyoxyethylene units, as shown in Supplemental Material, Figure S2A),
isosorbide monooleates, and sorbitan dioleates, indicating the presence
of Tween 80. The other highly abundant component of the CWAF
ethanol/water sub-fraction was DOSS. Initial investigation of the
extract in negative FS mode revealed an intense ion at m/z 421. A total
ion chromatogram performed in negative FS mode is shown in Supplemental
Material, Figure S2B, with the peak at 2.45 min largely representing the
[[M-H].sup.-] ion of DOSS. To confirm the presence of DOSS, a product
ion scan of m/z 421.1 was performed, and the resultant fragmentation
profile is shown in Supplemental Material, Figure S2B. Based on previous
reports, which indicate the presence of fragment ions m/z 81 and m/z 227
(Mathew et al. 2012; Ramirez et al. 2013), it was confirmed that DOSS
was abundantly present in the 50:50 ethanol:water sub-fraction.

Molecular modeling of COREXIT components binding to the PPAR[gamma]
ligand-binding domain. Molecular modeling was assessed using MOE
software (Molecular Operating Environment; Chemical Computing Group,
Inc.). Two PDB (Protein Data Bank; http:// www.wwpdb.org/) crystal
structures, 4EMA and 2HFP, constituting the human PPAR[gamma]
ligand-binding domain bound to different ligands, were compared. Using
MOE's superposition function, no significant differences were found
in the active site of PPAR[gamma] [RMSD (root-mean-square deviation)
< 2[Angstrom]]. Because it contained our positive control
Rosiglitazone (Rosi), 4EMA was chosen as the model. Ten compounds,
including some metabolites, were docked to PPAR[gamma]. Molecular
parameters were set for maximum energy minimization and Amber12 liquid
state. Five compounds that constitute COREXIT, a negative control
(17[beta]-estradiol), and a positive control (Rosi) were assessed and
virtually synthesized based on liquid state parameters at pH 7.4. The
run was set to 30 different poses, with an area for 30 refinements if
necessary. After parameters were set, a continuous run of all compounds
were docked, and descriptors of binding data were given in ascending
order related to E score. The E score gives binding efficiency in terms
of energy state, with lower E scores indicating higher affinity.
Compounds that had the capability to be cleaved by esterases were
assessed with and without the fatty acid chains.

In vivo bioluminescence imaging. The "repTOP PPRE-Luc"
mouse model [PPAR response element (PPRE)-luciferase mouse], containing
a PPAR response element-luciferase reporter transgene (El-Jamal et al.
2013) was used for in vivo bioluminescence imaging. C57BL/6J mice
(originally obtained from Charles River Laboratories) were bred in the
animal facilities at the Medical University of South Carolina (MUSC).
All mice were treated humanely and with regard to alleviation of
suffering, according to the Guide for the Care and Use of Laboratory
Animals (National Research Council 2011). Mice were housed up to five
per cage in ventilated air racks with ad libitum access to water and
irradiated rodent chow (Harlan 2018). Temperature was maintained between
21[degrees]C and 23[degrees]C, humidity maintained 40%, and a 12-hr
light:dark cycle. Five groups of three male mice (littermates age 5-6
weeks) were injected i.p. (intraperitoneally) at 0900 hours in the
animal housing facility with saline, 10 mg/kg Rosi (Rosiglitazone;
Cayman Chemical), or 50 mg/kg DOSS (Dioctyl sodium sulfosuccinate; Sigma
Aldrich). Five hours later mice were anesthetized with 2.5% isofluorane
(Sigma Aldrich) in air and injected i.p. with 150 mg/kg luciferin
potassium salt (Goldbio). An IVIS 200 bioluminescence imaging system and
Living image 4.3.1 software were used to quantify bioluminescence
according to the manufacturer's instructions (Caliper Life
Sciences). Specifically, mice were placed in the instrument and received
2% isofluorane in air through nosecones to maintain sedation during
image acquisition. PPRE-driven bioluminescence of live mice was
quantified using images acquired 10 min after luciferin injection with
1-min exposures. While mice were still sedated, they were sacrificed by
cervical dislocation, livers were dissected and washed in PBS and then
homogenized in lysis buffer, and luciferase activity was determined in
liver homogenates using the Luciferase Reporter Assay System and a
Veritas microplate luminometer. All animal procedures were approved by
the Institutional Animal Care & Use Committee, MUSC (Animal Welfare
Assurance #A3428-01).

TR-FRET PPAR[gamma] competitive binding assay. Competitive
time-resolved fluorescence resonance energy transfer (TR-FRET) (Toth et
al. 2012) binding assays were used to determine the affinity of DOSS for
PPAR[gamma] (SelectScreen Service; Life Technologies). For these assays
the donor fluorophore (terbium) on the receptor causes energy transfer
to the acceptor fluorophore (Fluormone Green) on the bound ligand,
resulting in emission at 520 nM. With increasing doses of ligand (e.g.,
DOSS), there is increasing displacement of receptor-bound Fluormone
Green-tagged positive control ligand and, hence, less signal. Known
concentrations of test and control ligands allow fluorescent emission
loss to be used to quantitate binding affinity. Terbium is excited with
a 340-nm filter and emits multiple peaks, the first of which (485-505
nm) overlaps with the maximum excitation peak of Fluormone Green. To
measure energy transfer to Fluormone Green without interference from
terbium, a 520/25-nm filter is used with a 100-[micro]sec delay and
200-[micro]sec integration.

Adipogenic differentiation assays. For triacylglycerol staining
assays to quantify adipogenic differentiation, 3T3-L1 preadipocyte cells
(Zenbio) were plated in 48-well plates at a density of 10,000 cells per
well in preadipocyte growth medium (PGM; DMEM/F12 supplemented as
described above) and grown until confluence. Two days postconfluence,
the media were changed to minimal induction media [MIM; PGM supplemented
with 62.5 nM dexamethasone, 0.125 mM IBMX (3-isobutyl-1-methylxanthine),
and 250 ng/mL insulin] and either different concentrations of DOSS (10,
20, 25, or 50 ppm) or 1 [micro]M Rosi. After 72 hr of induction, the
media was switched to PGM containing 1 pg/mL insulin. Cells were allowed
to differentiate for 3 more days (6 days total) and fixed with 4%
paraformaldehyde. Triacylglycerol staining with AdipoRed (Lonza) and
nuclear counter-staining with NucBlue (Hoechst; Invitrogen) was
conducting according to the manufacturers' recommendations.
Adipogenesis was quantified by mean relative fluorescent units of 42
fields per well (10x magnification) with five replicates used for each
treatment using a HERMES high content screening scanner (WiScan; IDEA
Bio-Medical Ltd.). Hoechst fluorescence was determined using excitation
390/18 nm and emission 440/40 nm (light intensity: 50%; exposure: 30
msec; gain: 30%), and AdipoRed was quantified using excitation 485/20 nm
and emission 525/30 nm (light intensity: 90%; exposure: 58 msec; gain:
30%).

mRNA expression via quantitative polymerase chain reaction (qPCR).
3T3-L1 cells were treated with test ligands as described above. After 72
hr of exposure to MIM supplemented with varying concentrations of DOSS
or Rosi, three wells of treatment were pooled from each of two
experiments for mRNA expression analyses. RNA was isolated using the
RNeasy Kit (Qiagen), following the manufacturer's instructions.
Gene expression was assessed in triplicate using 25 ng of RNA per qPCR
reaction and the iTaq Universal SYBR Green One-Step Kit following the
manufacturer's instructions (BioRad). Results were normalized to
the housekeeping gene Hprt. Data are expressed as a fold change compared
with the MIM-only control. Primer sequences for the queried genes can be
found in Supplemental Material, Table S1.

PPAR[gamma] ligand-binding assay optimization. We optimized a
rapid, sensitive, specific, and robust system for detecting PPAR[gamma]
transactivation activity in MC252 oil and COREXIT dispersant.
Considering that COREXIT is a mixture of solvents and surfactants that
might compromise membrane integrity and also that components of fetal
bovine serum (FBS) may have PPAR[gamma] agonist activity that would
obscure testing, we determined whether a PPAR[gamma] ligand-binding
domain-GAL4-UAS luciferase system (Forman et al. 1995) could be
conducted with cells under serum-free (SF) itions. HEK293T/17 cells were
transfected with plasmids encoding a yeast GAL4 DNA-binding domain-mouse
PPAR[gamma] ligand-binding domain fusion protein, a yeast UAS (GAL4
upstream activator sequence) driving firefly luciferase, and a
constitutively active Renilla luciferase transfection efficiency
control. The PPAR[gamma] agonist Rosi confirmed the accuracy of the
system for detecting PPAR[gamma] transactivation activity. As shown in
Figure 1, treatment of transfected cells with 0, 10 nM, or 100 nM Rosi
for 4 hr, 8 hr, or 18 hr resulted in time- and dose-dependent increases
in luciferase activity in both serum-containing (10% FBS) and SF
conditions. Notably, the response to Rosi under SF culture conditions
was more pronounced than serum-containing cultures at all time points
(Figure 1A-C). Conversely, treatment for 18 hr under SF conditions with
17[beta]-estradiol ([E.sub.2]) or all-transretinoic acid (RA) did not
increase luciferase activity, whereas Rosi treatment induced marked
luciferase activity (Figure 1D). These data demonstrate the sensitivity
and specificity of the PPAR[gamma] ligand-binding domain-GAL4-UAS
luciferase system. SF conditions and 18-hr treatments were chosen for
subsequent experiments aimed at identifying components of oil and
COREXIT bearing PPAR[gamma] transactivation activity.

PPAR[gamma] transactivation. To distinguish between PPAR[gamma]
activity originating from dispersant and from MC252 oil, several
mixtures of MC252 oil, with and without COREXIT, were prepared and
analyzed for PPAR[gamma] transactivation activity, including CWAF, WAF,
[C.sub.M]WAF, and COREXIT only (see "Materials and Methods").
Dose-dependent PPAR[gamma] activation was detected in CWAF,
[C.sub.M]WAF, and COREXIT dilutions but not in WAF (Figure 2). The CWAF
and [C.sub.M]WAF fractions comprise the aqueous fraction of the original
mixtures of oil and dispersant, and a portion of the amphipathic
compounds present in the original mixtures was expected to partition to
the organic phase. Also, the dose-dependent PPAR[gamma] activation by
COREXIT alone (no organic phase) substantially outstripped those of CWAF
and [C.sub.M]WAF; these results suggest that components of COREXIT were
responsible for the activity detected. To further investigate whether
oil might have contributed to PPAR[gamma] activation by the fractions,
an alternate solvent, DMSO, was used to prepare DWAF (DMSO
water-accommodated fraction), which lacks COREXIT. Although twice the
amount of DMSO was used to prepare DWAF than COREXIT in CWAF and in
[C.sub.M]WAF, no PPAR[gamma] activation was observed in any DWAF
dilutions tested (see Supplemental Material, Figure S1), further
implicating a COREXIT ingredient and not a component of oil as a
PPAR[gamma] activator.

CWAF fractionation and analysis. SPE was employed to separate CWAF
into fractions based on polarity and hydrophobicity; CWAF was
fractionated into 50:50 water:ethanol, methanol, DCM, and hexane soluble
fractions. The PPAR[gamma] transactivation system demonstrated
substantial PPAR[gamma] transactivation activity in the 50:50
water:ethanol fraction, whereas no activity was detected in the
methanol, DCM (dichloromethane), and hexane fractions (Figure 3). The
compounds present in the CWAF water:ethanol fraction were identified
using LC-MS and LC-MS/ MS as described in "Materials and
Methods." Analysis of the fraction in positive FS mode resulted in
> 200 unique target masses. Tween 80 (polysorbate 80) was concluded
to be a highly abundant component of this fraction based on manual
inspection and product ion scan profiles (see Supplemental Material,
Figure S2A). After investigation of the extract in negative FS mode,
DOSS was determined to be another highly abundant component of the CWAF
water:ethanol fraction (see Supplemental Material, Figure S2B). Mass
spectrometry analyses of the transactivation positive fraction argued
that relatively hydrophilic components of COREXIT and not MC252 oil were
responsible for the observed PPAR[gamma] transactivation activity.

Modeling of COREXIT components binding to PPAR[gamma]. Molecular
modeling was employed to predict which COREXIT components might bind to
PPAR[gamma]. Span 80, Tween 80, and DOSS were predicted to bind to the
PPAR[gamma] ligand-binding domain as shown by their low E scores,
whereas propylene glycol and 2-butoxyethanol were not (see Supplemental
Material, Figure S3A). Span 80 and Tween 80 have an ester bond that
could be cleaved by cellular esterases; once cleaved, neither cleavage
product is predicted to bind tightly (see Supplemental Material, Figure
S3A). For DOSS docking, the basic lysine 367 residue has a strong
hydrogen bond with a sulfonyl oxygen (see Supplemental Material, Figure
S3B). The a-carbon adjacent to the sulfonyl group of DOSS shows
potential for strong donation to a hydrogen of methionine 364.
PPAR[gamma] has multiple basic residues that allow pairing with the
acidic sulfhydryl group of DOSS, potentially allowing hydrogen bonding
between the ligand and receptor. Tween 80, along with other compounds
that exhibit low E scores, either have too large a fatty acid group to
effectively fit into the binding site of PPAR[gamma] or have a charge
that is too basic, resulting in higher binding scores (see Supplemental
Material, Figure S3A).

PPAR[gamma] transactivation activity of COREXIT components.
Collectively, the results above implicate a COREXIT component(s) in the
PPAR[gamma] transactivation observed in the CWAF prepared from MC252
oil. Mass spectrometry indicates that Tween 80 and DOSS are present in
the 50:50 water:ethanol fraction of CWAF, which exhibits activity in the
transactivation assay, and molecular modeling predicts that Span 80,
Tween 80, and DOSS can bind to the PPAR[gamma] ligand-binding domain.
Because molecular modeling is speculative, these compounds were tested
for PPAR[gamma] activity. Span 80 did not demonstrate PPAR[gamma]
transactivation activity even at concentrations much higher than
effective COREXIT alone dilutions (see Supplemental Material, Figure
S4A). Tween 80 had weak activity that was much too low to account for
the activity observed in CWAF and COREXIT (see Supplemental Material,
Figure S4B). Furthermore, a mixture of petroleum distillate [ICP
(inductively coupled plasma) solvent; CAS (Chemical Abstracts Service)
64742-47-8] and propylene glycol (PG), other major components of
COREXIT, did not demonstrate PPAR[gamma] transactivation activity (see
Supplemental Material, Figure S4C). In contrast, a simplified version of
COREXIT containing only ICP, PG, and DOSS demonstrated robust
PPAR[gamma] transactivation (see Supplemental Material, Figure S4D).
DOSS alone elicited dose-dependent increases in PPAR[gamma]-driven
luciferase expression in the low ppm range (Figure 4). Thus, DOSS has
PPAR[gamma] transactivation activity, whereas Span 80, ICP, and PG do
not. Because COREXIT 9500 is approximately 10% DOSS (Kujawinski et al.
2011), it is likely that the PPAR[gamma] agonist activity observed
following treatment with CWAF, [C.sub.M]WAF, or COREXIT alone is due to
DOSS.

DOSS PPAR[gamma] agonist activity in PPRE-luciferase transgenic
mice. To validate the PPAR[gamma] agonist activity of DOSS, PPRE
luciferase reporter mice were used as an in vivo model. Male littermates
age 5-6 weeks were injected with Rosi (positive control), saline
(negative control), or DOSS. Imaging from live mice 5 hr post-treatment
revealed marked increases in bioluminesence for both Rosi and DOSS
treatments and suggested that liver tissue was the main source of the
differential luciferase expression, with some expression in the skin of
Rosi- and DOSS-treated mice (Figure 5A). To confirm differential PPRE
activity in Rosi- and DOSS-treated hepatocytes, liver was dissected and
homogenized immediately following imaging and humane sacrifice. As
shown, treatment of PPRE-luciferase mice with 10 mg/kg Rosi increased
liver luciferase activity approximately 2-fold, whereas treatment with
50 mg/kg DOSS elicited about a 4-fold increase (Figure 5C),
demonstrating that DOSS is capable of activating PPAR-driven gene
expression in mice. Although PPAR[alpha], a major metabolic regulator,
is the predominant PPAR isoform in the liver, PPAR[gamma] is also
expressed in hepatocytes. PPRE activation in the liver opened the
possibility that DOSS may activate other PPAR isoforms or RXR[alpha]
(retinoid X receptor alpha). Transactivation assays using the human LBDs
(ligand-binding domain) indicated that 4 ppm DOSS activated PPAR[gamma]
about 3 fold, whereas activation of PPAR[alpha] was only about 1.2 fold
and nondetectable for the human RXR[alpha] LBD (see Supplemental
Material, Figure S5A,B,D). Of note, DOSS activated PPAR[beta]/[delta]
about 8 fold at 4 ppm, which is under further investigation (see
Supplemental Material, Figure S5C).

TR-FRET assays of DOSS affinity for PPAR[gamma]. The structure of
DOSS is shown in Figure 6A. The predicted PPAR[gamma] receptor-binding
affinities are shown in Supplemental Material, Figure S3B. TR-FRET
assays were used to measure the affinity of DOSS for PPAR[gamma]. As
shown in Figure 6B, DOSS binds to the human PPAR[gamma] ligand-binding
domain with a [K.sub.D] of 1,380 nM. This is a binding affinity
comparable to the pharmaceutical PPAR[gamma] agonist pioglitazone
([K.sub.D] = 1,310 nM) and the endogenous ligand arachidonic acid
([K.sub.D] = 1,340 nM), as determined in the same assay (Singh et al.
2008).

Increases in general health and longevity over the past century are
tributes to our knowledge of the scientific basis of good health. These
impressive and hard-won gains are now threatened by an obesity epidemic
(Stewart et al. 2009). Poor nutrition and lifestyle are well-established
drivers of obesity. Additional contributors might include
"obesogens," compounds that alter metabolism and fat cell
production, as indicated by cell culture and animal model studies (Grun
and Blumberg 2006; Janesick and Blumberg 2011). Maternal-fetal obesogen
exposure studies in animal models suggest that obesogens might
exacerbate multiple lifelong health issues (Daftary and Taylor 2006;
Janesick and Blumberg 2011; Suzuki et al. 2007) and might exacerbate
obesity in future generations (Li et al. 2012; Tracey et al. 2013).
Still, sound evidence of direct effects in humans remains to be
determined; it is unclear whether environmental obesogens, singly or in
combination, act through known metabolic and/or adipogenic mechanisms
(e.g., via PPAR[gamma]) to cause clinically significant outcomes in
humans, such as obesity, type 2 diabetes, insulin resistance syndrome,
and polycystic ovary syndrome. Obesogens that are most prevalent in the
environment, including those in widely used dispersants, consumables,
and personal care products would be a good focus of such attention.

Here we have identified a novel likely obesogen, DOSS, as evidenced
by PPAR[gamma] transactivation activity in a transfected cell line,
activation of PPRE in transgenic reporter mice, and induction of
adipogenesis in preadipocytes in vitro. DOSS affinity to human
PPAR[gamma] receptor was also shown to be comparable with the
pharmaceutical pioglitazone and an endogenous ligand arachidonic acid.
In addition to being a principal component of COREXIT oil dispersant
used during DWH remediation, DOSS is also used in many consumer goods,
including laxatives, household cleaning products, deodorants, hair
coloring, and nail polishes (DHHS 2014; EWG 2015a). That DOSS is
"generally recognized as safe" and is a common additive in
flavored drinks means that its use may continue to be widespread,
creating the likelihood of long-term exposures [EWG 2015b; U.S. Food and
Drug Administration (FDA) 1998]. Significantly, DOSS itself (Colace) is
prescribed as a laxative for pregnant women, in which there is a 38%
prevalence of constipation, the treatment with which could possibly
affect fetal development (Jewell and Young 2001). Also, fetuses and
neonates can be at greater risk to food-borne contaminants, so groups
studying seafood contaminant levels suggest that the FDA revise the
levels of concern for these compounds for pregnant women and children
(Rotkin-Ellman et al. 2012). Although environmentally relevant DOSS
exposures are not yet known, oral laxative use in pregnant women of up
to 500 mg/day (88.5 kg in an average term pregnancy) would be expected
to be within an order of magnitude of the dose of DOSS used in our
initial mouse studies, which resulted in significant PPRE activity in
vivo (Figure 5). Clearly, in vivo oral or topical dosing studies in
animals at doses that are environmentally relevant are warranted to
fully substantiate and understand the direct and long-term impacts of
such subtoxic level exposures of this likely obesogen (Buonsante et al.
2014).

Conclusion

The results of this study indicate that the major COREXIT
dispersant component DOSS is a potential obesogen. This work indicates
that DOSS might be a compound that negatively affects health and that
further investigation is warranted. The subsequent identification of
metabolites and biomarkers of DOSS exposure and biological consequences
of exposure will aid in assessing its contribution to obesity and
related health concerns.

(1) Marine Biomedical Sciences Program, and (2) Department of
Pathology and Laboratory Medicine, Medical University of South
Carolina, Charleston, South Carolina, USA; (3) Department of Chemistry,
Rollins College, Winter Park, Florida, USA; (4) Department of
Pharmaceutical Sciences, Medical University of South Carolina,
Charleston, South Carolina, USA; (5) Center of Excellence on
Neurodegenerative Diseases, University of Milan, Milan, Italy; (6)
Department of Obstetrics and Gynecology, Medical University of South
Carolina, Charleston, South Carolina, USA; (7) National Oceanic and
Atmospheric Administration, and (8) National Institute of Standards and
Technology, Charleston, South Carolina, USA; (9) Department of
Pediatrics and Neonatology, Medical University of South Carolina,
Charleston, South Carolina, USA

Caption: Figure 1. PPAR[gamma] transactivation activity in a
GAL4-UAS system using serum-free conditions. HEK293T/17 cells were
transfected and exposed to the PPAR[gamma] agonist Rosi under
serum-containing (10% FBS) and serum-free (SF) conditions in triplicate,
and luciferase activities were measured following exposure for (A) 4 hr,
(B) 8 hr, and (C) 18 hr. Data in A-C are normalized to the 10% FBS
control within each time point. Assay sensitivity is greatly enhanced
under SF conditions. (D) HEK293T/17 cells were transfected and exposed
to estrogen ([E.sub.2]), all-trans-retinoic acid (RA), or Rosi under SF
conditions, and luciferase activities were measured, demonstrating
ligand-specific responsiveness of the system. Data in D are normalized
to the untreated control. RLU, relative light units. Data are expressed
as mean [+ or -] SD; n = 3 per group (* p < 0.05 vs. control).